A neural circuit is composed of a population of neurons that are interconnected by synapses and carry out a specific function when activated. Thus, to identify a neural circuit, both the synaptic connections and the func- tional relevance of each neuron in the circuit must be examined. To date, few methods are available to study these two aspects of a neural circuit simultaneously. In the current proposal, this problem will be addressed using a new class of trans-synaptic techniques. Current trans-synaptic techniques enable the labelling of postsynaptic elements of first-order neurons without using driver lines. However, the ability to discriminate the function of each neuron in the circuit and the ability to track higher-order neurons remains lacking. A Drosophila thermosensory system that controls rapid warmth avoidance will be used as a model to demonstrate how a modified trans-synaptic technique can sparsely label both second- and higher-order neurons in a single neural circuit and determine their functional relevance to a specific behavioral response. The FLP/FRT recombination system will be used to sparsely label second- and higher-order neurons. Behavioral assays will be used to dis- criminate the functions of each labelled neuron. In Aim 1, the current trans-synaptic technique will be combined with the FLP/FRT recombination system and behavioral assays to sparsely label second-order neurons that are necessary and sufficient for a specific behavioral response. Aim 2 will use the same strategy as Aim 1 to sparsely label neurons that control discrete functions and express the trans-synaptic techniques in second-order neurons to track higher-order neurons. Therefore, in Aim 2, higher-order neurons that are necessary and sufficient for a specific behavioral response will be sparsely tracked. The expected outcome of this proposal is development of a tool that can (1) sparsely label synaptically interconnected second- and higher-order neurons without using driver lines, and (2) determine the function of labelled neurons. The tool will be able to be readily applied to many other Drosophila neural circuits, where the synaptic connections and the functional relevance of each neuron will be simultaneously identified. Moreover, it has the potential to provide an alternative for labelling mammalian circuits.